For various physiological conditions, such as cancer, infectious diseases, physiological disorders, and so forth, detection and monitoring may be based, in part, on the analysis of a biological specimen from the patient. For example, a sample may be analyzed to detect the presence of abnormal numbers or types of cells and/or organisms that may be indicative of a disease or disorder. Various types of microscopy may be employed for such analysis. Further, various stains and staining protocols may be employed as part of this analysis to allow visualization of different structures, chemicals, or environments that might aid in detection or diagnosis of a disease or disorder.
For diagnostics in cancer biology, multiplexed applications analyze tumor samples on a single cell level by studying the expression of more than 175 different proteins and disease markers per cell. Multiple stains can be added to a tumor slice; tumor antibodies directed to proteins of interest are measured and quantified by fluorescence. Because multiple stains and markers are measured in a single test, the amount of provided sample tissue need not be an issue. Using a single slide saves time, uses significantly less tissue, and provides a far more consistent result.
Every cell gets addressed on a digital map creating a graphic representation of protein expression in the tumor tissue. The data can be matched and compared with known protein expression profiles in other tumors to provide information on tumor characteristics and prognosis. Pathologists use software to identify expression patterns and perform cluster analysis on protein profiles to identify correlations and understand tumor behavior.
To facilitate analysis of such pathology or histology samples, automated microscopy systems have been developed that automate various aspects of the image acquisition process. In particular, digital optical microscopes may be used in such automated systems and provide a digital image output for each acquisition. Certain such systems employ scanning microscopes where a sequence of displaced images are acquired and associated together (e.g., “tiled” or “stitched” together) to form a composite of the sample region of interest. For example, in the context of pathology and histology imaging operations, tissue sample slides undergo imaging to acquire digital images of small adjacent or overlapping areas at high magnification and/or resolution. The adjacent or overlapping images may then be joined or associated to form a larger image that may be navigated on a digital display device. In this manner, a composite or mosaic image of the sample is generated, displayed, and navigated by a reviewer.
A complicating factor in the image generation and review process may be attributed to protocols where a sample undergoes multiple staining operations. In such instances, each staining step is associated with removing the slide from the microscope stage, treating the sample to remove any existing stain, applying the next stain, and replacing the slide on the microscope stage for imaging of the sample with the new stain. The act, however, of removing and replacing the slide on the microscope stage generally results in the slide being at a slightly different position for each round of imaging. As a result, corresponding images from each round of imaging may not be aligned. Further the composite images generated for each round of imaging may also be misaligned with respect to one another. As a result, analyses or comparisons conducted having images acquired using different stains are difficult or otherwise inhibited.
For the multiplexing (“MultiOmyx”) process, it is critical that images taken from a single slide at different points in time all be registered to within a single pixel. As the slides may have physically left the microscope between these points, to allow for various operations such as staining and bleaching to take place, both mechanical alignment and software based image registration are necessary to align images of the slide. In loading the slide back in the microscope, the slide is inserted in a slide holder. Issues arise, however, when the slide is inserted incorrectly in the slide holder (i.e. rotated or offset). This can occur both because of user error and/or because of an accumulation of debris in either the holder or on the edge of the slide.
For imaging rounds other than the first round, an existing approach ensures that a slide is loaded into a holder and placed in a microscope well aligned with the slide position from the initial imaging round. This approach relies on comparing images of a reference channel (e.g. typically using DAPI (4′,6′ diamino-2-phenylindole.2HC1 staining) which stains the nucleus and does not change from round to round, e.g. between the current round and the initial round. By detecting changes in the image placements, a set of linear transformations are determined to relate the current slide position to the initial slide position. While this works well forsubsequent rounds, for the initial imaging round no other set of images are available for comparison; the initial imaging round is the baseline. This leaves the process susceptible to errors stemming from a misloaded slide in this initial imaging round. For example, operators change and multiple people are moving and replacing the slide during the process, such operators changing shifts in the clinical setting and attempting to keep the process as synchronous as possible.                Furthermore, techniques that currently exist to ensure subsequent imaging rounds proper align with respect to the first round are lacking. No methods to date have allowed for the detection of alignment failures in the first round. Later rounds depend upon this initial placement of a slide in the initial round. When errors do occur, later rounds can only be imaged if the slide is misloaded in the same way on later rounds as positioned on the first round. This proves very difficult as there are many ways to load a slide incorrectly as opposed to one way to have it loaded correctly.        
For exemplary purposes, and not limitation, in studies of Hodgkin's Lymphoma in MultiOmyx directed tests in clinical settings, several tests have failed due to slide placement failure. These failures have included where slides have failed through the process after the second round of imaging 1% or less of cases) due to misalignment on the first round of imaging. In these cases, a second slide had to be utilized to be cut from a tissue block and sent through the MultiOmyx process since the failure could not be detected until the second round after which irreversible processing had already occurred on the slide. As MultiOmyx is positioned as a technology with advantages when tissue supply is limited, there is no opportunity to cut new slides when a first round alignment fails. Additionally, when tissue is available, running a second slide through the process adds both delays and cost to completion of the analysis. By detecting this misalignment during the initial imaging round, correction can be made by a manual repositioning of the slide or by folding in the alignment information and digitally correcting the proceeding acquisition for the observed alignment error.
Most previous attempts have involved trying to make a mechanically precise slide holder. Many alternative designs have been used, but are still susceptible to either gross human error or fowling of the slide itself. For example, even with a complicated mechanical mechanism to push a slide against a reference corner, if a barcode is misapplied to a slide and overhangs, the slide will still be pushed against a corner, though with the reference point including the barcode. When the barcode is replaced in later processing rounds (i.e. currently in the MultiOmyx process, barcodes are added and removed before staining) and when the slide is reloaded, it will sit at a different reference point (as the overhanging barcode will no longer be there). Additionally, for applications to date outside the field of MultiOmyx, it is not important that a slide be precisely repositioned in an imager for later imaging. Thus, in most cases it is ok if there is some discrepancy in slide alignment. Furthermore, work in detecting coverslip edges to limit imaging to under the coverslip does not address the above described issues either.
Thus, a need exists to have a technique to detect misloading of a slide in the initial imaging round. The technique will address appropriate loading of the slide by characterizing an angle and an offset of the slide, while further allowing comparison with a bounded value. The invention will address these needs in order to address optical quality and performance of the imaging technique, and comply with ongoing clinical processes.